Validation of reference genes for gene expression studies in the emerald ash borer (Agrilus planipennis)

The Emerald ash borer (EAB, Agrilus planipennis Fairmaire) an exotic invasive insect pest has killed millions of ash trees (Fraxinus spp.) across North America and threatens billions more. We validated six A. planipennis reference genes (actin, ACT; beta tubulin, beta-TUB; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; ribosomal protein, RPL7; translation elongation factor 1 alpha, TEF-1 alpha; and ubiquitin, UBQ) using geNorm, Nonrifinder and BestKeeper for accurate determination of target messenger RNA levels in gene expression studies. The stability of the six reference genes was evaluated in different larval tissues, developmental stages and two treatments of A. planipennis using quantitative real-time polymerase chain reaction. Although there was no consistent ranking observed among the reference genes across the samples, the overall analysis revealed TEF-1 alpha as the most stable reference gene. GAPDH and ACT showed least stability for all the samples studied. We conclude that TEF-1 alpha is the most appropriate reference gene for gene expression studies in A. planipennis. Results obtained can be applicable for transcript profiling in other invasive insect pests. Further, these validated reference genes could also serve as the basis for selection of candidate reference genes in any given insect system post-validation.